The long range goal of the proposed work is to define the molecular components and mechanisms mediating V. cholerae colonization and virulence protein secretion to the point where there is sufficient knowledge to intelligently incorporate this information into improved cholera vaccine strategies and antimicrobial therapies designed to inhibit these events. Most of the proposal involves analysis of the molecular mechanisms by which TCP (toxin correlated pilus) is formed and mediates intestinal colonization. TCP is the major factor required for colonization by V. cholerae, with tcpA mutants of all epidemic serogroups and biotypes showing a 5 log decrease in colonization ability. A number of properties attributable to TCP that potentially contribute to colonization will be examined. These include a collaborative effort to solve the pilin crystallographic structure, study of the role of bacterial aggregates formed upon autoagglutination, search for specific receptor functions, study of the mechanisms of TcpA-mediated serum resistance, and further definition of potential immunogenic domains of TcpA. The practical application of incorporating this knowledge into the use of TcpA peptide vaccines will also be tested. In addition, because of the discovery that the cholera toxin genes are located on a lysogenic transducing phage that utilizes TCP as a receptor, the PI will seek tcpA mutations for incorporation and improved safety in live, oral vaccine strains that still allow colonization functions of TCP, but abolish the phage receptor or uptake function. Some steps in the process by which the pilus is built are also linked and related to the process of toxin and other virulence determinant secretion and represent potential targets for antimicrobial intervention. The PI will further examine two aspects of pilus biogenesis in detail; that of the processing of prepilin to mature pilin by the TcpJ type 4 prepilin peptidase, and the mechanism by which the TcpG (DsbA) periplasmic disulfide bond oxidoreductase interacts with its target substrates. These studies will involve mutational analyses to further define active sites involved in these events in conjunction with continuing collaborative efforts that have led to solving the crystallographic structure of TcpG, and will now include structures incorporating peptide substrates. Lastly, the use of the MshA pilin for surface antigen display in V. cholerae heterologous carrier vaccine strains will be examined. This pilus is immunogenic, yet dispensable for V. cholerae colonization, thus making it a candidate for pilin replacement by other type 4 pilins or partial substitution with heterologous epitopes.